Help Reading Sonication Gels! - (Dec/15/2006 )
I was wondering if any of you could help interpret my trial DNA shearing on these gels. I used a Microson XL sonicator. I sonicated for 10 sec 8 X with a resting interval of 1.5 minutes.
I started off sonicated at 4 different power settings: 2.5, 5, 10, 15. From reading a substantial amount of other posts, I know that lower power settings are better - but I thought I would try some higher ones to what happened. No foaming at 10 or 15 but they do seem less effient.
Important note: I used a cold block (so everything took place cold and on ice) for this round. The tube didn't move b/t sonication rounds and the probe stayed in the sample during the resting interval.
Then I sonicated at 2.5, 3, 4, 5. (****The two wells with setting 3 are from the same tube - when loading the gel sample from first 3 lane floated out, so I loaded another.) Same sonicator, pulse number duration, waiting time - BUT I didn't have the block so I held the tube out of ice at the top during the pulse and then put it back in ice during resting interval.
I am thinking that this difference might play a role in how the smears look a little different.
Also there is a fatter band sitting at the 1 kb band almost all samples. Is this unsonicated DNA? How do I get over this threshold? I need my DNA to be around 500-200 bp for my primers.
Any help, input, interpretation is greatly appreciated.
Have you RNase treated these samples. If not, I would think that the smear you see from about 500bp and smaller is likely RNA (I could be wrong but this is what I see in my own gels). In any case, it's easy to figure out by adding RNase and seeing if the smear goes away. The band you see around 1kb is, I think, your DNA. Is there any reason why 1kb is unacceptable. We routinely have an average fragment size around 1kb but can see differences in factor binding with as little separation as 200bp.
I have not treated with RNase - but that is probably what those smears around 500-100 bps are. Didn't even think about that. Thanks for the input. I will treat this samples with RNase and see what the gel looks like.
As for the PCR - we are looking at p27 sp1 response element with the control being 1 kb away at the p27 distal element. That's why we want the shearing to be less than a kb. Does it look like the fragments are sitting between the 1kb and 600 bp range to you?
In our experience, the resolution we get is often better than the average fragment length would suggest. This is probably because, due to differences in chromatin architecture, some areas are more easily fragmented than others. I would guess that, within the region upstream of the transcription start site, the fragmentation would be better than in other intergenic sites or within hetrochromatin (just a a guess; I haven't seen anything to confirm this). If your chromatin isn't very precious, you might just try a ChIP and run a few primers around the region you're interested in. If you can differentiate between a positive control site (where you expect your factor to bind) and a negative control site a few hundred bp away, then I'd say your resolution is likely just fine.
Sounds like a good plan! Thanks for all the help and input - it makes my life with ChIP a little bit easier.